1. Field of the Invention
This invention generally relates to thermostable enzymes capable of degrading (hydrolyzing) cellulose at high temperatures, and the incorporation of nucleic acids coding for one or more of such enzymes into a host, and, more particularly, a host that produces or is composed of cellulosic material.
2. Background of the Invention
Cellulose is a polysaccharide consisting of a linear chain of several hundred to over nine thousand β (1→4) linked D-glucose units [formula (C6H10O5)n]. Cellulose is the most abundant organic compound on earth, making up about 33 percent of all plant matter, about 50 percent of wood, and about 90 percent of products such as cotton. In nature, cellulose is present as part of the lignocellulosic biomass of plants, which is composed of cellulose, hemicellulose, and lignin. The carbohydrate polymers (cellulose and hemicelluloses) are tightly bound to the lignin, by hydrogen and covalent bonds.
Many highly desirable products are derived from lignocellulosic biomass. In particular, much interest has recently been focused on recapturing the saccharide building blocks locked in plant biomass for biofuel production. For example, fermentation of plant biomass to ethanol is an attractive carbon neutral energy option since the combustion of ethanol from biomass produces no net carbon dioxide in the earth's atmosphere. Further, biomass is readily available, and its fermentation provides an attractive way to dispose of many industrial and agricultural waste products. Finally, plant biomass is a highly renewable resource. Many dedicated energy crops can provide high energy biomass, which may be harvested multiple times each year.
One barrier to the production of products from biomass is that the cellulosic polymer has evolved to resist degradation and to confer hydrolytic stability and structural robustness to the cell walls of plants. This robustness or “recalcitrance” is due largely to extensive intermolecular hydrogen bonding between cellulose polymer chains. Some organisms, notably fungi, bacteria, and protozoans, but also some plants and animals, have evolved the ability to digest cellulose. In vivo cellulose breakdown typically entails the cooperative interaction of several cellulases, enzymes that catalyze the cellulolysis (hydrolysis) of cellulose. Several different kinds of cellulases, which differ structurally and mechanistically, are known, and some of these have been isolated, characterized and used to break down cellulose in vitro. General categories of cellulases include: endo-cellulases (endoglucanases), which randomly hydrolyze internal bonds to disrupt the crystalline structure of cellulose, thereby exposing individual cellulose polysaccharide chains; and exo-cellulases (exo-processive-endoglucanases), which cleave 2-4 units from the ends of the exposed chains produced by endocellulases to produce tetrasaccharides or disaccharides such as cellobiose. Two major types of exo-cellulases are known, one of which works processively from the reducing end, and one of which works processively from the non-reducing end of cellulose. A third major type of cellulase is cellobiase or beta-glucosidase, which hydrolyses exo-cellulase products such as cellobiose into individual glucose monosaccharides.
Typically, the digestion of cellulose is carried out at temperatures approaching 100° C. because, at high temperatures, intermolecular hydrogen bonds are disrupted and recalcitrant cellulose polymers become accessible to the cellulase enzymes. Therefore, cellulases used commercially in such processes must be able to withstand very high temperatures, preferably for extended periods of time.
There is an ongoing need to identify, isolate and characterize cellulases, especially thermally stable cellulases, for use in the enzymatic hydrolysis of cellulose. Of particular interest is the development of groups or systems of cellulases that include enzymes with endo-cellulase, exo-cellulase and beta-glucosidase activity, the enzymes in the system acting in concert to carry out the complete hydrolysis of cellulose to glucose at high temperatures.